Abstract
Microbial influences on host cells depend upon the identities of the microbes, their spatial localization, and the responses they invoke on specific host cell populations. Multimodal analyses of both microbes and host cells in a spatially resolved fashion would enable studies into these complex interactions in native tissue environments, potentially in clinical specimens. While techniques to preserve each of the microbial and host cell compartments have been used to examine tissues and microbes separately, we endeavored to develop approaches to simultaneously analyze both compartments. Herein, we established an original method for mucus preservation using Poloxamer 407 (also known as Pluronic F-127), a thermoreversible polymer with mucus-adhesive characteristics. We demonstrate that this approach can preserve spatially-defined compartments of the mucus bi-layer in the colon and the bacterial communities within, compared with their marked absence when tissues were processed with traditional formalin-fixed paraffin-embedded (FFPE) pipelines. Additionally, antigens for antibody staining of host cells were preserved and signal intensity for 16S rRNA fluorescence in situ hybridization (FISH) was enhanced in poloxamer-fixed samples. This in turn enabled us to integrate multimodal analysis using a modified multiplex immunofluorescence (MxIF) protocol. Importantly, we have formulated Poloxamer 407 to polymerize and cross-link at room temperature for use in clinical workflows. These results suggest that the fixative formulation of Poloxamer 407 can be integrated into biospecimen collection pipelines for simultaneous analysis of microbes and host cells.
Highlights
Investigating host–microbe interactions may reveal the mechanisms underlying how changes in the gastrointestinal microbiome are related to colorectal cancer (CRC)
Microbiota–host interactions are implicated in a range of disorders, including CRC
Human CRC-associated stool microbiota transplanted into GF or carcinogen-treated mice exacerbates colon epithelial cell proliferation and the tumor-associated immune response[45]
Summary
Investigating host–microbe interactions may reveal the mechanisms underlying how changes in the gastrointestinal microbiome are related to colorectal cancer (CRC). Increased abundance of Fusobacterium nucleatum, Bacteroides fragilis, and Escherichia coli have been associated with human CRC1–3 These species are hypothesized to promote tumorigenesis and/or progression of CRC through upregulation of oncogenes, modification of intestinal mucus, or damage to host DNA2,4,5. The inner, striated mucus layer of the colon is largely sterile in healthy hosts[6] Perturbations of this tightly regulated spatial organization have been linked to both inflammatory bowel diseases and CRC12–15. Mucosa-associated biofilms have been identified in ~50% of sporadic CRC patients, 100% of familial adenomatous polyposis (FAP) patients, and 13% of healthy individuals. They are enriched in CRCs from the ascending colon[3]. Bacterial isolates from CRC or FAP biofilms have been shown to promote colonic tumor progression in genetically susceptible mice[13,15]
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